1
Q
why study proteins
A
- everything in body is either a protein or made by one
- proteins catalyze every process in cell
- genetic diseases caused by proteins
- infectious diseases dependent on proteins
- targets of drugs
- transmit info
- antibodies protect
2
Q
functions of proteins determined by
A
- polymer length
- aa composition
3
Q
molecular medicine
A
- can’t go directly from genes to disease and treatment yet
- need to go through aa sequence, protein structure to find function, which can lead to cause of disease, drug design, and prevention to understand disease and treatment
4
Q
proteome
A
-content of proteins within the cell at any given time (more complex than the genome)
5
Q
Tipranivir
A
- prescribed anti-HIV drug
- Asp26 (one from each monomer) are residues responsible for nuclephilic attack on the substrate
- non hydrolyzable
6
Q
alzheimers
A
- characterized by extensive deposits of misfolded proteins (amyloid fibers) in the brain
- associated with cell death and loss of brain function
- main component of these is a 42-residue fragment from the Alzheimer precursor protein (APP)
- APP is normally cleaved to a 40-residue fragment
- two extra AA enough to convert normal soluble protein to a sticky peptide that builds up in the brain
7
Q
Concepts from lecture
A
- proteins are the only polymers that spontaneously fold from an unstructured noodle to a specific 3D shape (except RNA- but not nearly as many structures)
- the only thing different about the noodle and 3D shape is bond angles
- patterns of atomic bumping in polypeptides favor certain combinations of bond rotations and prohibit others
- allowed and disallowed bond angles determine types of structure that can form
8
Q
protein structure
A
-not rocks, quite unstable (deltaG=0-10kcal/mol)
-constantly fold and unfold
-up to ~40% contain regions on intrinsic disorder
-many diseases caused by improper folding or degradation
-
9
Q
phi and psi 1
A
- each peptide has phi,psi angle combo
- determine twists and turns chains take
- alpha helix (or beta sheet) residues have similar phi, psi combos
- alpha closer to 0-scrunched
- beta closer to 180- almost fully extended
- angle seen between atoms when sighting down central bond psi=carbon-carbon, phi=carbon-nitrogen
10
Q
Ramachandran plot
A
- plot of allowed angles of phi and psi
- only a small fraction allowed (dark grey)
- only allows certain structures to form
- plots arise simply from how atoms are connected (bond length, angle, and hard-sphere repulsions
- glycine is a BAMF-more options
11
Q
properties of amino acids
A
- hydrophobic
- hydrophilic
- unique (pro, gly, cys)
- protein structure can be understood by the binary code of polar on the outside, polar on the inside
- function in many instances boils down to a few amino acids
- amino acid substitutions are common, effect can be significant or not
12
Q
Asp pKa
A
4
13
Q
Glu pKa
A
4
14
Q
His pKa
A
6.5
15
Q
Cys pKa
A
8.5
16
Q
Lys pKa
A
10
17
Q
Arg pKa
A
12
18
Q
carboxy terminus pKa
A
4
19
Q
amino terminus pKa
A
8
20
Q
hydrophobic-aliphatic (most)
A
- alanine
- valine
- leucine
- isoleu
- proline
21
Q
aromatic, sulfur containing side chains, less hydrophobic
A
- phenylalanine
- tyrosine
- tryptophan
- methionine
22
Q
pKa and ionization
A
- acid dissociation constant
- low pKa=acidic, binds H loosely
- high pKa=basic, binds h tightly
- pKa is pH at which half the ionizing groups are protonated, half are deprotonated
- pHpKa, low H in solution, draws off H
23
Q
pKa cont
A
if pKa of group is 4, at pH 7 will be deprotonated (because likes to be proton donor (acid) and there aren’t that many around). at pH 3, will be protonated because there are lots of protons around
-if pH is 4, half the groups would be protonated and half wouldn’t be
24
Q
negatively charged side chains (at pH7)
A
pKa’s low- like to give protons up (at pH7), acidic, only protonated at pHs lower than pKa
- aspartic acid
- glutamic acid
- cysteine (pKa 8.5, but sulfur likes to give H to form SS bonds)
25
positively charged side chains (at pH7)
- pKas high, like to keep protons (at pH7), only deprotonated at pHs higher than pKa
- histidine
- lysine
- arginine
26
histidine
- can accept and donate protons at physiological pH
- only molecule that ionizes near physiological pH
- used as catalytic residue by enzymes in acid-base catalysis
- 5 membered ring is imidazole ring
27
structure can be used to change pKa (Asp)
- local environment can lower or raise pKa by >4 units
- very important for enzymes
- Asp is neg at physiological pH
- adding excess H will put it back on- enough so that pH is 4
- if Asp is near pos AA, really wants to give up its H, pKa decreases (becomes more acidic, binds H looser)
- if Asp near neg AA, keep its H more than usual, pKa increases, becomes less acidic, binds H tighter
- same if its near a greasy blob
28
polar uncharged side chains-hydroxyl
- pKa-16-keeps its H except in extreme conditions
- serine
- threonine
- electronegative atoms
29
polar uncharged side chains-amide
- asparagine
- glutamine
- electronegative atoms
- more hydrophilic than ser or thr
30
unique side chains
- Glycine-lack of side chain-flexible
- Proline-cis peptide bond favors kinks, turns. no H-bond donor on peptide bond
- Cysteine-thiol group can oxidize to SS
31
covalent properties of polypeptides
Backbone:
- properties are dominated by the peptide bond
- simple bonding and steric considerations encourage formation of certain types of structures
- peptide groups are necessary for 3D structure, but not sufficient
AA side chains
- gives proteins their individuality
- chemical interactions and shape of side chains determine structure
*3D structure cannot be predicted by AA chain alone
32
protein folding
- non-covalent interactions
- well folded is only 5-15 kcal/mol more stable than unfolded (2-3 kcal/mol)
- structure is dynamic
- bond rotations is how it folds
- accumulation of small favorable non-covalents that overcome unfavorable loss of rotational entropy
Forces:
- electrostatic
- dipolar-salt bridges and other interactions
- hydrogen bonding
- van der waals
- hydrophobic effect
33
Electrostatic interactions
- all follow coulomb's law (e=q1q2/r^2D)
- D is dielectric constant-polarity of local environment-vacuum is 1 water is 80
- salt bridges and other interactions
- maximize charge-charge and charge-water interactions
- ion pairs, Asp, Glu, Lys, Arg and His often found on protein surface-solubility of globular proteins in water
- charged groups rarely on interior
- ion pairs also help in DNA binding proteins, provide counter charge to substrate to help binding
34
salt bridges
- strongest electrostatic forces
- frequently observed on protein surfaces
- rarely seen in interior
35
buried charge
- rhodopsin
| - only Lys residue in transmembrane helices, forms Schiff base with the chromophore, 11-cis retinal
36
ATP synthase and buried charge
- F0 proton channel in ATP synthase- proton translocation channel is formed by helical subunits that sits in the membrane and rotates.
- helices non-polar except single Glu-proton binds Glu and rides like merry go round
- kink in helix by Pro residue three positions down from Gly
37
Dipolar interactions-weak electrostatic
- 2 charges separated by distance
- dipole arises from difference in electronegativity
- no net charge on molecule
- dipole moment- mu-(excess charge) X (separation)
- partial positive and negative without formal net charge
- electronegative atoms responsible
- common is peptide bond
- antiparallel arrangement is energetically favorable
- occurs in beta sheets
- alpha helices are parallel-offset by charged side chains
38
hydrogen bonding
- special electrostatic interaction
- 2 electronegative atoms compete for same H
- oxygen, nitrogen, sulfur
- in proteins, all donors are bonded to acceptors and vise versa
- strong because of coulombs law- peptide carboxylate (full charge) strongest
- straight stronger than kinked
- can also occur b/n charged groups
39
proteins as polymers
- compact 3D shape
- specific function
- highly efficient
- folds on its own
- other polymers do not fold
40
amino acids
- building blocks
- alpha carbon-amino,cooh, and side chain
- central carbon is chiral but only L enantiomer synthesized and incorporated into proteins
41
peptide bond
- planar (no rot around DB) and trans (steric clash)
- two resonance forms
- 60% left (DB O) 40% right (DB N)
- C-N bond 40% DB character
- rotations about the bonds to and from the central carbon
42
planarity
- reduces bond rotations
- two rotatable bonds per residue
- rotatable bonds define backbone and are called phi (C-C) and psi (C-N)
- look at pictures for phi and psi
43
phi and psi 2
- tell us about conformation of polypeptide backbone
- only 10-20% of allowed conformations are found in nature
- rotatable bonds
- 180- extended conformation
- 0-compaction
44
van der Waals forces
- mutually induced dipole (transient)
- present between all atoms
- net energy bonus as a result of favorable electrostatic interactions
- approximated by lennard-jones potential
- proteins fold to maximize van der Waals energy (tightly packed cores)
- optimal distances in core for maximum interactions
- no empty space
45
hydrophobic effect
- principle glue that holds proteins together
- major DF for interactions with hormones, nucleic acids, and other proteins
- tendency of certain molecules to interact with themselves and not with water
- non-polar/hydrophobic groups
- lack charges, dipoles, polar groups, and H bonding groups
46
hydrophobic effect and water
- hydrophobic molecules do not have attraction for themselves or repulsion for water-hydrophobic effect is due to water
- water has unusually high attraction for itself due to polarity and H-bonding properties (donor and acceptor)
- non-polar groups can't H bond, and to make up for this, water changes its structure in the vicinity of dissolved hydrophobic molecules
- structure is called clathrates-ice like
- energetically unfavorable and origin of effect
47
clathrates
- when non-polar molecules and water interact (in aq solns), water forms highly ordered structures around the molecule
- form H bonded icebergs around the non-polar surface
- decrease in entropy (can rotate more freely in bulk) is responsible for the tendency of non-polar molecules to cluster together, reducing non-polar surface exposed to water
48
secondary structure
- alpha helix, beta sheets, beta turns
- short, simple, repeating
- stabilized by interactions between residues close in sequence
- can occur in short peptides
49
tertiary structure
- longer polypeptides associate with one another to form these
- overall 3D conformation
- stabilized by short range as well as long range contact between residues distant in sequence
- usually required for the protein to function although not often sufficient (need quaternary)
50
quaternary structure
- two or more individual subunits that function as a whole
| - all foldings are stabilized by electrostatic, h bonding, vdF and hydrophobic
51
alpha helix
- most frequently observed
- average protein contains 31% helix
- characterized by specific H bonding patters between the peptide NH of i and the peptide CO of i+4
- peptide backbone coils to make a rod, the side chains stick out
- side chains tilted toward N terminus-can't rotate freely
- periodicity of 3.6 residues
- chains of every 4th residue lie on approx the same face of the helix
- 5.4 angstroms per turn
- average 12 residues long
52
3.6 residue repeat
- favorable backbone dihedral angles
- near optimal H bond geometry
- good VdW contacts between backbone atoms
- other types of helices possible but uncommon
53
stabilization of alpha helix
- backbone-backbone
- backbone-side chain
- side chain-side chain
54
backbone- backbone interactions in alpha helix
- extensive network of NH and CO bonds formed b/n peptide groups 4 residues apart
- H bonds have optimal geo and aligned parallel to helix axis
- tightly packed, good VdW
55
backbone-side chain interactions in alpha helix
- peptide bonds have polarity
- 0.4 e units of pos on N and neg on COOh
- results in permanent dipole
- all aligned and form macro-dipole
- therefore overall pos at N terminus and overall neg on C terminus
- place corresponding residues near termini to stabilize helix
56
side chain-side chain interactions in alpha helix
- 3.6 residue repeat means side chains of i and i+4 are closest
- in contact with each other via H bonds, electrostatic, hydrophobic, and VdW
57
helix former AA
- alanine
| - no side chain means default backbone conformation is helical
58
strong helix breakers
- Proline-no NH to H bond
| - Glycine-flexible
59
medium helix breakers
- beta branched or bulky
- Valine
- Threonine
- Tryptophan
- Phenylalanine
- lose much rotational freedom (entropy) in helix
60
helix indifferent
- long, straight chains
- arginine
- lysine
- glutamate
- lose less rotational freedom (Alanin loses none)
61
Myoglobin (and other alpha helices)
- one side of helix usually faces in and the other faces out (non-polar and polar)
- tightly associated with cell membranes
- membrane anchors consist of several helices containing mostly non-polar residues-interact with interior of lipid bilayer
- disrupted by detergents
62
beta sheets
- average protein contains 28% beta sheets
- made up of two or more beta strands
- polypeptide chain is straight and nearly completely extended
- H bonds are formed between peptide groups of each strand
- pleated appearance
- parallel or antiparallel
- parallel H bonds slightly bent, so antiparallel more likely
63
why beta sheets?
- favorable backbone dihedral angles
- phi,psi is about 140 degrees, chain is nearly fully extended
- rise is 3.5 angstroms
- periodicity is 2-side chains are 2 apart
- straight H bonds between strands
- interaction between adjacent strands is tertiary and called amyloid
- pattern of hydrophobic amino acids stabilizes
- frequently amphipathic
64
interconversion between helices and sheets
- polypeptides can form both
- implicated in disease
- myoglobin- can form amyloid- which causes disease
65
reverse beta turn
- 1/4 of protein structure
- several types
- much sequence variability
- glycine required-relieves steric clashes
- proline preferred-covalent bondbetween the side chain and main chain puts a kink in the backgone
- other positions are solvent exposed and polar residues
- polypeptide has to reverse directions in a globular protein
- tight turns-3-4 residues
- stabilized by H bonding
66
irregular protein structure
- random coil- but not truly random-wouldn't crystallize
- generally only random in the sense that it is not periodic
- usually has specific structure
- surface loops are critical to function
- loops give proteins individuality
- found on protein surface and are comprised of mostly hydrophilic residues
- flexible and tether globular domains together
- frequently form the binding site with another protein or substrate
67
protein loop
- stretch of polypeptide that is neither alpha helix, beta sheet, of reverse turn
- lack regular H bonded conformation and are not secondary structure
68
motifs
- small functional units that are part of larger structures
- short stretches of secondary structures
- not usually stable by themselves
- used in molecular recognition
- helix turn helix
69
helix turn helix
- proteins recognize specific sequences of DNA
- found in all kingdoms of life
- recognition alpha helix and a support alpha helix, connected by a turn
- recognition helix sits in major groove of DNA and binds to specific sequences of nucleotides
- structure of recognition helix maintained by side chain-side chain contacts between it and the support helix
- helices held together by a small hydrophobic core consisting of non-polar side chains
- i,i+3(4) spacing of hydrophobic residues make sure the HTH fold is achieved
70
zinc finger
- cysteins and histines
- binds DNA weakly
- TF proteins use 2-40 repeats (of zinc finger chain) to bind DNA
71
domains
- stable, semi independent units of structure
- residues within the domain interact more with each other than with the residues outside of it
- stable in isolation
- continuous stretches of polypeptide that are linked together by flexible loops, making it east to identify them
72
coiled coil domain
- extremely stable
- fibrous proteins and TFs
- heptad repeat
- a-f
- hydrophobic at a and d (1 and 4)
- e and g usually opposite charge than a and d (5 and 7)
- interact in helical wheel projection
- a and d are hydrophobic glue
- e and g provide electrostatic interactions
- DNA binding, protein-protein recognition, mechanical orce transductions, viral penetration
- hemagglutin
73
GCN4
- DNA binding domain is coiled coil
- stabilizing half has heptad repeats
- binding part is only helical and stable when bound
- intrinsically disordered
- see slide on pg 153
74
hemagglutin
- stalk domain is triple-stranded coiled coil
- H=hemagglutin, N=neraminidase- account for differences in flu
- Spanish flu was hybrid from recombination of 2 or more
- HA is molecular harpoon
- Flu enters, binds to cells via head domain of HA. endocytosed into cell (in capsule), when pH hits 5 from proton pumps, conformation change-extended loop folds to alpha helix
- loop in tail domain and links tops of coiled coil helices to shorter alpha helices near bottom of coiled coil helices-coiled coil lengthens
- head domain splays open, newly lengthened coiled coil thrust out and penetrate vacuolar membrane (tip is hydrophobic)
- fusion of two membranes
- similar to HIV (receptor binding), SARS, ebola
- protein drugs don't work well because they are degraded-D peptide can bind tighter and has longer life
- look at picture for HIV
75
pore-forming proteins
- small (<50kD)
- self associate and insert into PM of host
- pokes a hole, causes leakage and cell death
- anthrax, pneumonia, meningitis, cholera
- alpha hemolysin
- hydrophobic/philic interactions shape structure and function
MCP Unit 2
flashcards
Decks in class (10)
# Cards
